Serine proteinase from Cucurbita ficifolia seed; purification, properties, substrate specificity and action on native squash trypsin inhibitor (CMTI I).
A proteinase was purified from resting seeds of Cucurbita ficifolia by ammonium sulfate fractionation and successive chromatography on CM-cellulose, Sephacryl S-300 and TSK DEAE-2SW (HPLC) columns. Inhibition by DFP and PMSF suggests that the enzyme is a serine proteinase. The apparent molecular mass of this enzyme is ca. 77 kDa. The optimum activity for hydrolysis of casein and Suc-Ala-Ala-Pro-Phe-pNA is around pH 10.5. The following peptide bonds in the oxidized insulin B-chain were hydrolysed by the proteinase: Phe1-Val2, Asn3-Gln4, Gln4-His5, Cya7-Gly8, Glu13-Ala14, Ala14-Leu15, Cya19-Gly20, Pro28-Lys29 and Lys29-Ala30. The proteinase is more selective towards the native squash seed trypsin inhibitor (CMTI I) and primarily cuts off only its N-terminal arginine. The inhibitor devoided of the N-terminal arginine residue is still active against trypsin.
Dryjanski
M
M
Instytut Biochemii, Uniwersytet Wrocławski, Wrocław, Poland.
Otlewski
J
J
Polanowski
A
A
Wilusz
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014361:Trypsin Inhibitors / Q000378:metabolism*
D007328:Insulin; D010455:Peptides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C031809:trypsin inhibitor, Cucurbita sp.; D012697:Serine Endopeptidases